Zi Shen decoction inhibits non-small cell lung cancer progression by regulating macrophage polarization via the gut microbiota

OBJECTIVE: To investigate whether Zi Shen decoction (ZSD) inhibits the growth of non-small cell lung cancer (NSCLC) tumors by modulating macrophage polarization through gut microbiota-mediated mechanisms. METHODS: UHPLC-MS/MS identified ZSD components, and network pharmacology predicted its anti-tumor targets. In vitro, 3-(4,5-dimethylthia-zoly-2)-2,5-diphenyltetrazolium bromide (MTT) assay evaluated ZSD toxicity on RAW264.7 macrophages, and the nitric oxide (NO) content assay detected the level of NO in RAW264.7 macrophages, while Reverse transcription-polymerase chain reaction (RT-PCR) detected its effects on IL-4/IL-13-induced M2 polarization markers. In vivo, 16S rRNA sequencing analyzed gut microbiota; ELISA, RT-qPCR, and immunofluorescence assessed TAM polarization in tumors. An ABX-induced pseudo-sterile model verified gut microbiota's role. KEY FINDINGS: UHPLC-MS/MS analysis identified 92 compounds in ZSD, while network pharmacology predicted 233 core targets associated with tumor necrosis factor-α and T cell receptor signaling pathways. In vitro experiments demonstrated that ZSD exhibited no significant cytotoxicity toward RAW264.7 cells but effectively modulated macrophage polarization. In vivo, ZSD significantly suppressed transplanted tumor growth, altered the composition of gut microbiota, particularly increasing the abundance of Akkermansia, and promoted TAMs polarization toward the M1 phenotype while inhibiting M2 polarization. Notably, ABX abrogated both the anti-tumor effects of ZSD and its regulatory impact on TAMs polarization. CONCLUSIONS: Gut microbiota is the key to ZSD reshaping macrophage polarization to exert anti-NSCLC tumor growth effect in mice.